WO1995021849A1

WO1995021849A1 - Process for separating double-stranded/single-stranded nucleic acid structures

Info

Publication number: WO1995021849A1
Application number: PCT/EP1995/000445
Authority: WO
Inventors: Helge Bastian; Simone Gauch; Metin Colpan; Petra Feuser
Original assignee: Qiagen Gmbh
Priority date: 1994-02-11
Filing date: 1995-02-08
Publication date: 1995-08-17
Also published as: US20020081619A1; US6180778B1; US20030224389A1; JP3256238B2; DK0743950T3; EP0743950B1; ATE302208T1; US6946250B2; EP0743950A1; DE59509941D1; US7074916B2; DK1146049T3; DE59511013D1; ATE210671T1; EP1146049B1; JP4036625B2; EP1146049A3; EP1146049A2; JP2002187897A; JPH09508638A

Abstract

A chromatographic process is disclosed for separating nucleic acid mixtures into their double-stranded and single-stranded fractions. All nucleic acids are simultaneously adsorbed in a mineral substrate, then separated by fractionated elution into double-stranded and single-stranded nucleic acids, or double-stranded and single-stranded nucleic acids of a sample are selectively adsorbed in a mineral substrate. Also disclosed are solutions and a kit for carrying out this process.

Description

Process for the separation of double-strand / single-strand nucleic acid structures

The present invention relates to a method for the chromatographic separation of nucleic acid mixtures into their double- and single-stranded nucleic acid components, in that total nucleic acids are simultaneously absorbed on a mineral support and the separation into double-stranded nucleic acid and single-stranded nucleic acid is then carried out by fractional elution or by selectively absorbing double-stranded nucleic acid or single-stranded nucleic acid of a liquid sample onto a mineral carrier as well as solutions and kit for carrying out the method according to the invention.

Preparation of nucleic acids, both RNA and DNA, has become increasingly important. For example, the biological sources from which the RNA or DNA is to be isolated are opened up, for example by mechanical influences or chemical influences, such as treatment with detergents, etc. Thus, cell digestion to obtain the nucleic acid is usually followed by a cesium chloride density degree. centrifugation or extraction with phenol. Although these methods are suitable for isolating nucleic acids, they have disadvantages which make their handling more difficult. For example, the cesium chloride density gradient centrifugation requires the use of time-consuming and cost-intensive ultracentrifugation, whereas the handling of phenol does not appear to be harmless for reasons of occupational health and safety.

In the past, there has been no lack of attempts to simplify the isolation of nucleic acids. DE 36 39 949 AI, DE 40 34 036 AI or DE 41 39 664 AI are concerned, for example, with improvements in nucleic acid purification by means of chromatographic methods while avoiding expensive apparatus-related processes such as high pressure liquid chromatography (HPLC). Although these methods are already an advance over ultracentrifugation or phenol extraction, for example, they are technically relatively complex and labor-intensive. Since several cleaning steps often have to be carried out one after the other for fractionation, the processing of small sample amounts in particular B. problematic due to loss of substance.

EP 0 389 063 A2 also relates to a method for isolating nucleic acids. The source containing nucleic acids is disrupted in the presence of chaotropic ions and then treated with a material that adsorbs nucleic acids under these conditions. Diatomaceous earth or other mineral carriers containing silicon oxide are mentioned as such a material. It is possible to isolate RNA and DNA and RNA and ssRNA simultaneously using the method mentioned in EP 0 389 063 A2. However, a desirable fractionation of the nucleic acids bound to the silicon dioxide carrier into DNA and RNA fractions is not achieved. RNA can then be broken down by adding RNAse so that the DNA remains.

Gillespie et al. disclose in U.S. Patent No. No. 5,155,018 a process for the isolation and purification of biologically active RNA from biological sources containing RNA, DNA and other cell contents. The RNA-containing source is brought into contact with particles which are silica gel-containing materials such as finely divided glass. The binding buffer, from which the RNA is adsorbed on the material, is solutions containing acidified chaotropic salts. Under these conditions, RNA but not DNA is bound to the silica. The use of acidified chaotropic buffers has the disadvantage that that during the acidification of guanidinium thiocyanate (GTC) -containing binding buffers there is a risk of hydrocyanic acid formation and special precautionary measures must therefore be taken. Furthermore, the DNA is destroyed by the action of acid. In addition, DNA purification from the authentic sample cannot be carried out by this method.

Little describes in U.S. Patent No. 5,075,430 a process for the purification of plasmid and other DNA, both single-stranded and double-stranded, by immobilizing the DNA on diatomaceous earth in the presence of a chaotropic agent, whereupon the DNA is eluted with water or with a buffer with a low salt content. The method cannot purify DNA / RNA.

M. A. Marko et al. in "Analytical Biochemistry" 121, pages 382 to 387 (1982) describe a process for the large-scale isolation of highly purified plasmid DNA using the alkaline extraction and binding to glass powder. Fractionation and separate purification of RNA and DNA from a single sample is not described.

Follow-up reactions follow the crude preparation of the nucleic acids. These subsequent reactions place certain requirements both on the isolation procedure and on the purity and integrity of the isolated nucleic acids. In particular, if subsequently enzymatic amplification reactions such as PCR (polymerase chain reaction), LCR (ligase chain reaction), NASBA (nucleic acid sequence-based amplification) or 3SR (self-sustained sequence replication) are used, the preparation should be used of the nucleic acids without the risk of cross-contamination of other samples, and the isolated nucleic acids should be free from interfering cell components and / or metabolites. The enzymatic Due to their specificity and sensitivity, amplification of DNA (eg PCR) or RNA (eg RNA-PCR) is gaining importance not only in basic research but also increasingly in the medical field for diagnostic purposes. For example, for the detection of nucleic acid sequences from the smallest amounts of cells and / or tissues or biopsy materials or for the detection of viral nucleic acids from blood or plasma. For these applications, in addition to the requirements mentioned, the yield and reproducibility of the nucleic acid isolation process are extremely demanding.

A technical problem on which the invention is based is to specify a method with which it is not only possible to purify RNA and DNA from biological samples such as cell lysates and tissue lysates separately from one another but originating from the same sample, but generally double-stranded from single-stranded nucleic acids . The process should work as inexpensively as possible, for example by using inexpensive unmodified separation materials. The method should also be suitable for sample preparation for diagnostics and should be compatible with various amplification methods. Furthermore, the disadvantages mentioned in the discussion of the prior art should be avoided.

Surprisingly, the technical problem on which the invention is based is solved by a method having the features of patent claim 1 in its alternative methods 1.1 to 1.4. Subclaims 2 to 11 relate to preferred embodiments of the method according to the invention, claims 12 to 21 solutions for use in the method according to the invention or the use of these solutions and claim 22 relates to a compilation containing the components necessary for the method according to the invention. The process according to the invention for the fractionation of double-stranded and single-stranded nucleic acid structures from biological sources is described in detail in the following process alternatives.

The sample to be separated (single- and double-stranded) containing sample is treated with at least one mineral carrier, the treatment conditions being adjusted by a corresponding aqueous mixture of salts, in particular chaotropic substances and substance containing alcohol groups, so that predominantly the single-stranded Nucleic acid fraction is adsorbed on a first mineral carrier, while the double-stranded nucleic acid is not adsorbed. The emerging double-stranded nucleic acid can then be processed further using methods known per se. The single-stranded nucleic acid adsorbed on the first mineral carrier is eluted after washing steps which may have been carried out under conditions of low ionic strength or with water. The non-adsorbed double-stranded nucleic acid that has been collected can be further purified by e.g. the fraction is then adjusted to such conditions by means of a corresponding aqueous mixture of salts, in particular chaotropic substances, and substances containing alcohol groups that the double-stranded nucleic acid can be adsorbed on a second mineral carrier and, after washing steps which may have been carried out, becomes less elutable under conditions Ionic strength or with water.

In a second embodiment of the method according to the invention, for the separation of single-stranded nucleic acid and double-stranded nucleic acid, the treatment conditions are set such that alkaline earth metal complexing substances are present in the solution in the absence of substances containing alcohol groups, the single-stranded nucleic acid is not adsorbed on the first mineral support and can be separated from the rest of the sample. The separated single-stranded nucleic acid can then be processed further using methods known per se. However, the double-stranded nucleic acid predominantly binds to the first mineral carrier and, after washing steps which may have been carried out, can be eluted under conditions of low ionic strength or with water. The double-stranded nucleic acid thus obtained can then be processed further using methods known per se.

The non-adsorbed single-stranded nucleic acid which has been collected can then be adjusted to such conditions, in particular by adding substances containing alcohol groups, that the single-stranded nucleic acid can then be adsorbed on a second mineral carrier. and, after washing steps which may have been carried out, can be eluted under conditions of low ionic strength or with water.

If the treatment conditions are set so that wetting agents, detergents or dispersants are present in the solution in the absence of alcohol-containing substances, the single-stranded nucleic acid is not adsorbed on a first mineral carrier under these treatment conditions and can therefore be separated from the rest of the sample and further are processed. However, the double-stranded nucleic acid predominantly binds to the first mineral carrier and, after washing steps which may have been carried out, can be eluted under conditions of low ionic strength or with water. The eluted double-stranded nucleic acid can then be processed further using methods known per se.

The non-adsorbed, collected single-stranded nucleic acid can then be adjusted to such conditions, preferably by adding substances containing alcohol groups, that the single-stranded nucleic acid is then on a second mineral carrier is adsorbable and, after washing steps which may have been carried out, becomes elutable under conditions of low ionic strength or with water.

Another embodiment of the method according to the invention ensures the fractionation of jointly bound single-stranded nucleic acid and double-stranded nucleic acid. The treatment conditions are adjusted by a corresponding aqueous mixture of salts, in particular chaotropic substances and substances containing alcohol groups, such that the total nucleic acid from single-stranded nucleic acid and double-stranded nucleic acid is adsorbed on a mineral carrier, followed by fractionation of the double-stranded / single-stranded nucleic acid by selective elution of the double-stranded nucleic acid by treatment with a solution of reduced ionic strength and concentration of a substance containing alcohol groups or elution of the single-stranded nucleic acid by means of a solution containing an alkaline earth metal complexing substance and / or a wetting, washing or dispersing agent and one or several types of salt (s), especially chaotropic substances. In the first case the single-stranded nucleic acid then remains bound to the carrier, whereas in the second case the double-stranded nucleic acid remains bound to the mineral carrier. The eluted fraction in each case can then be processed further using methods known per se.

The treatment conditions with substances and salts containing alcohol groups, in particular chaotropic substances for separating the nucleic acids, are set according to the invention on the basis of the following physico-chemical principles, which are formulated here for the first time. 1 shows the binding of single-stranded / double-stranded nucleic acid using the example of single-stranded RNA and double-stranded DNA. The RNA / DNA binding from a tissue lysate to a mineral support is described depending on the concentration of a substance containing alcohol groups (here ethanol) and a chaotropic substance (here GTC). Provided that the concentration of one of the substances, alcohol or chaotropic substance, is constant, it must be established that, with a high alcohol concentration and / or quantity of chaotropic substance, both types of nucleic acid (RNA / DNA) are bound to the mineral carrier. If the concentration of one or both substances (alcohol or chaotropic reagent) falls below a certain value, then none of the nucleic acid binds to the mineral carrier to any appreciable extent. In between, RNA and DNA bind surprisingly so differently to the mineral support that this can be used to separate the nucleic acids. Thus, starting from cells, after lysis of the cells with a high concentration of chaotropic substances, subsequent addition of a substance containing alcohol groups or a mixture of substance containing alcohol groups and water or buffer, concentrations of chaotropic substance and alcohol groups Substance adjusted so that a selective binding of the RNA is achieved while the DNA remains in the breakthrough. In the example according to FIG. 1, one would choose a concentration of 1.75 M GTC and 30% by volume ethanol in order to achieve a separation of RNA from DNA by fractional binding.

On the other hand, under conditions of high concentration of substance containing alcohol groups and / or high concentration of chaotropic substance, simultaneous binding of single-stranded nucleic acid and double-stranded nucleic acid to the mineral carrier can be achieved, and by reducing the concentration of substance containing alcohol groups and / or chaotropic substance, the desorption of the double-stranded nucleic acid is initially initiated. The single-stranded nucleic acid remains bound and elutes when the concentration of one or both substances is reduced still further. In the example according to FIG. 1, concentrations of 1.75 M GTC and 45 vol% ethanol would be selected in order to achieve binding of the total nucleic acid. As exemplified in Example 8, one would choose a concentration of 0.3 M GTC and 10 vol% ethanol for the selective desorption of the DNA.

It is therefore possible to separate the RNA and DNA by adsorption on a mineral support, or else first to adsorb the total nucleic acid onto the mineral support and to selectively elute the single-stranded nucleic acid or double-stranded nucleic acid.

If necessary, washing steps can also be carried out before the elution of the respective nucleic acid (single-stranded nucleic acid or double-stranded nucleic acid).

The elution is then carried out under conditions of low ionic strength or with water. The nucleic acid initially desorbed from the mineral carrier is then adjusted by increasing the ionic strength and / or the concentration of substances containing alcohol groups such that the double-stranded nucleic acid or single-stranded nucleic acid is adsorbed on a second mineral carrier and, after washing steps which may have been carried out, eluted under Low ionic strength conditions or with water.

Since nucleic acids, for example, can also be adsorbed onto mineral carriers in sodium chloride / ethanol mixtures and can be eluted under conditions of low ionic strength or with water to assume that the salt solutions used in the process according to the invention do not necessarily have to contain chaotropic salts, but that each salt solution can be used in combination with a substance containing alcohol groups.

The method according to the invention advantageously enables the processing of small amounts of samples, ensures simple and safe handling while avoiding precipitation steps. Furthermore, the method according to the invention can be carried out in a low-cost and personnel-intensive manner and can easily enable the processing of a large number of samples simultaneously. Because of its versatility and ease of handling, the method is also suitable for automation.

According to the invention, double-strand / single-strand nucleic acid structures can be separated from sources containing nucleic acid structures with the method. As sources that can include nucleic acid structures to be separated, there are e.g. sources mentioned in claim 7 in question. These are cell cultures, tissues of all kinds, body fluids such as blood, plasma, serum, urine, faeces, microorganisms such as bacteria, viruses such as cytomegalovirus, HIV, hepatitis B, hepatitis C, hepatitis δ virus, plants, Plant parts, embryos, seedlings, fruits or nucleic acids-containing mixtures after enzymatic reactions such as in vitro transcription and / or cDNA synthesis and / or reverse transcription with subsequent polymerase chain reaction (PCR).

Cells are preferably first disrupted in an aqueous lysis system, which contains chaotropic substances and / or other salts, in the simplest case by adding the cells to them. If necessary, the process of disintegration can be accelerated by mechanical action. Then the sample treated in this way, depending on the problem, which nucleic acid type is to be separated from the other, as described in process steps 1.1 to 1.4 according to claim 1, is further processed.

Due to the nature of their cell walls, some of the starting materials mentioned, such as bacteria, cannot be lysed directly in aqueous systems containing chaotropic substances. These starting materials must therefore be pretreated before they can be used in the process according to the invention, for example with lytic enzymes.

Systems for lysing the sources containing the nucleic acid are preferably solutions of chaotropic substances in a concentration of 0.1 to 10 M. The chaotropic substances used are, in particular, salts such as sodium perchlorate, guanidinium hydrochloride, guanidinium isothiocyanate / guanidinium thiocyanate, Na - Trium iodide, potassium iodide and / or combinations thereof in question.

Aqueous solutions containing salts such as sodium chloride, lithium chloride, potassium chloride, sodium acetate, magnesium chloride in a concentration of 0.1 to 10 M or urea in corresponding concentrations of 0.1 to 10 M and / or combinations of these substances can also be used as aqueous systems for Lysis or binding of the sources containing the nucleic acid can be used.

The substances containing alcohol groups are preferably lower aliphatic alcohols having 1 to 5 carbon atoms, such as methanol _/ ethanol, isopropanol, butanol and pentanol. They are preferably used in a concentration of 1 to 90% by volume. The mineral carrier preferably consists of porous or non-porous metal oxides or mixed metal oxides, silica gel, materials which consist predominantly of glass, such as unmodified glass particles, glass powder, quartz, aluminum oxide, zeolites, titanium dioxide, zirconium dioxide with a particle size of the mineral carrier material of 0.1 μm to 1,000 μm and a pore size of 2 to 1,000 μm. The porous or non-porous carrier material can be in the form of loose fillings or can be designed as filter layers in the form of filter layers made of glass, quartz or ceramic and / or a membrane in which silica gel is arranged and / or as particles or fibers from mineral carriers and fabrics are made of quartz or glass wool and latex particles with or without functional groups or frit materials made of polyethylene, polypropylene, polyvinylidene fluoride, in particular ultra high molecular weight polyethylene, high density polyethylene.

In particular, ethylenediaminetetraacetic acid (EDTA) or EGTA can be used as the alkaline earth ion-binding substance and a sarcosinate can be used as wetting, washing or dispersing agent.

If desired, the nucleic acids obtained according to the invention can be purified by further chromatographic methods such as anion exchange chromatography.

In the method according to the invention, in particular RNA can be separated from double-stranded nucleic acid (DNA) as single-stranded nucleic acid. If DNA is single-stranded, this DNA can then also be separated from double-stranded DNA and also from double-stranded RNA.

The solutions used in the method according to the invention are also the subject of the present invention. According to the invention, lysis buffers and / or binding buffers are used in particular aqueous solutions containing 0.5 to 8.0 M guanidinium isothiocyanate / guanidinium thiocyanate and / or guanidine hydrochloride, 0 to 50% ethanol and / or isopropanol.

An aqueous solution containing 0.1 to 3 M guanidinium isothiocyanate / guanidinium thiocyanate and / or guanidine hydrochloride together with 1 to 30% by volume ethanol and is used as the solution for washing out or eluting nucleic acids bound to the mineral carrier / or isopropanol in question.

The aqueous solution system which can be used to bind double-stranded nucleic acid to mineral supports is an aqueous solution containing 1 to 5 M guanidine isothiocyanate and / or 1 to 8 M guanidine hydrochloride with 0.1 to 5% sarcosinates or 5mM to 200mM EDTA into consideration. A solution containing 1 to 5 M guanidinium thiocyanate and / or 1 to 8 M guanidinium hydrochloride, 5 mM to 200 mM EDTA or EGTA is also suitable for binding double-stranded nucleic acid.

The combination of components according to the invention for carrying out the method in a kit has, in particular, hollow bodies suitable for through-flow, in which the mineral carrier (s) is (are) arranged in the form already described above. The mineral carrier can be in bulk, which is fixed between two devices or in the form of membranes which are arranged in the hollow body. Furthermore, solutions can be contained in the kit or the components for the assembly of the solutions in concentrated form. From this, the user can then produce the required solutions in the necessary concentration.

Another advantageous component of the compilation is a device for homogenizing the solution on cell lysates. A particularly preferred device for homogenizing is proposed in the international patent application PCT / EP 95/00037. This device essentially consists of at least two porous layers, the porous layers having a decreasing pore size as seen in the direction of flow through the layers. As the cell lysate passes through the decreasing pore sizes, the viscous solutions of the cell lysate are homogenized.

The invention is explained in more detail in the following examples.

Materials and methods

I. Silica support materials

Silica support materials were used in membrane form or as suspended particles.

1 . 1 . Silica membranes

Two layers of a silica membrane (e.g. glass fiber filter from Whatman) were fixed in a centrifuge chromatography column ("spin column") as described in P 43 21 904. The standard protocol "spin procedure" (cf. 4.1) was used for membrane-shaped carrier materials.

1 .2. Silica particles

Various silica particles (e.g. from Merck, Darmstadt and Sigma, St. Louis) were used as a 50% suspension in the lysis buffer used (see 3.1). The average particle diameter was 0.5 to 50 μm depending on the material. The standard protocol "batch procedure" (cf. 4.2) was used. 2. Sources containing nucleic acid

2.1 tissue

The tissues used for the preparation were frozen in liquid nitrogen immediately after removal and stored at -70 ° C. Alternatively, fresh tissue can be used.

2.2. plants

Leaves were ground to a fine powder in a mortar under liquid nitrogen and used directly for preparation or stored at - 70 ° C.

2. 3. Cell culture

After the harvest, cells were washed twice with PBS and pelleted. Aliquots with a corresponding number of cells (determined by counting in a thoma chamber) were used fresh for the preparation or stored at −20 ° C.

Alternatively, adherently growing cells can be washed in the culture dish and lysed directly in the culture dish by adding the respective lysis buffer (see 3.1).

2nd 4th plasma

ACD blood was centrifuged at 3,000 xg for 10 min, the supernatant was removed and centrifuged again as above. The supernatant obtained after the second centrifugation was aliquoted and stored at -70 ° C. 2. 5. Bacteria

Cultures were inoculated with an overnight culture and grown to an OD ₆₀₀ of 0.5-0.8. Aliquots with the corresponding number of cells (1 OD ₆₀₀ = 10 ⁹ cells / ml) were pelleted and the cell pellets were stored at −20 ° C. or used fresh for the preparation.

3 • Reagents

3rd 1 . Lysis buffer

Ll 4.5 M GTC, 25 mM NaCitrate Ph 7.5, 0.7% β-mercaptoethanol

(MSH)

L2 4.0 M GTC, 25 mM NaCitrate pH 7.5, 0.7% β-MSH

L3 5.0 M GTC, 50 mM TRIS / HCl pH 7.0

L4 3.5 M GTC, 25 mM NaCitrate pH 7.5, 1% β-MSH

L5 2.5 M GTC, 25 mM NaCitrate pH 7.5, 1% ß-MSH, 30% ethanol

L6 8.0 M GuHCl, 20 mM MOPS pH 7.0, 0.7% β-MSH

L7 3.0 M GTC, 25 mM NaCitrate pH 7.5, 1% β-MSH

L8 4.0 M GTC, 50 mM TRIS / HCl pH 7.5, 1% Sarkosyl

L9 4.0 M GTC, 50 mM TRIS / HCl pH 7.5, 25 mM EDTA

3 .2. Binding reagent

Bl ethanol

B2 n-butanol

B3 isopropanol

B4 70% ethanol in water

B5 5.9 M GTC 3rd 3rd Wash buffer

Wl 2.0 M GTC, 25 mM TRIS / HCl pH 7.5, 30% ethanol

W2 4.0 M GTC, 40 mM TRIS / HCl pH 7.5, 20% isopropanol

W3 1.0 M GTC, 25 mM TRIS / HCl pH 7.5, 20% ethanol

W4 5.0 M GuHCl, 15 mM MOPS pH 7.0, 37% ethanol

W5 0.5 M GTC, 25 mM TRIS / HCl pH 7.5, 10% ethanol

4. Standard protocols

4th 1 . "spin procedure"

1) Add the lysis buffer (see 3.1.) To the source containing nucleic acid and homogenize completely using a handheld homogenizer

2) Add binding reagent (see 3.2.) To set the respective binding conditions

3) Pipette the lysate onto the spin column and centrifuge through the membrane of the spin column at 10,000 rpm for 15 seconds; if the volume of the lysate exceeds the filling volume of the spin column, repeat this binding step

4) If necessary, further process or discard the column opening

5) Pipette 700 μl washing buffer (cf. 3.3.) Onto the spin column and centrifuge as described in 3) in order to remove contaminating cell components

6) Wash the membrane-bound nucleic acids salt-free twice with 700 μl 80% ethanol in water, proceeding as in 5)

7) centrifuge the spin column for two minutes at maximum speed in order to completely remove ethanol 8) 50 to 100 ul Pipette C heated water to 80 ° directly on the membrane of the spin column and 1 minute at maxi¬ ^painters' speed centrifuge to elute the nucleic acids; Repeat the elution step if necessary.

4 .2. "ba tch procedure"

2) Add binding reagent (see 3.2.) To set the respective binding conditions

3) Add 50 μl silica suspension (50% in the lysis buffer) and incubate for 10 minutes to bind the nucleic acids at room temperature, whirl vigorously several times (vortex)

4) Centrifuge for 15 seconds at 10,000 rpm in a table centrifuge in order to pellet the silica material

5) Pipette off the supernatant and, if necessary, further work up or discard

6) Add 700 μl wash buffer (cf. 3.3.) To the pellet, stir vigorously (vortex) until the pellet is completely resuspended and centrifuge as in 4)

7) Wash step 6) repeat twice with 700 μl 80% ethanol in water to wash the silica material salt-free

8) Dry the pelletized silica material for 10 minutes at 56 ° C with the lid open

9) Add 50 to 100 μl of water, completely resuspend the pellet by vigorous stirring (vortexing) and incubate for 10 minutes at 56 ° C, stirring vigorously several times (vortexing); Repeat this elution step if necessary

10) Centrifuge for 1 minute at maximum speed and transfer the supernatant to a new reaction tube. 5. Electrophoretic methods

The isolated nucleic acids were analyzed on ethidium bromide stained agarose gels. For this purpose, 1.2% formaldehyde or 1.2% 1 x TBE gels were made.

Formaldehyde gels were shaken in water for 3 to 4 hours after the run and then overnight in 10 μg / ml RNase A in order to digest the RNA and thus make the DNA more visible. TBE gels were RNase digested without prior equilibration.

The examples described below illustrate the implementation of the method according to the invention. All nucleic acids isolated after this were analyzed electrophoretically and quantified photometrically. The OD 260/280 value for all eluates was between 1.7 and 2.0.

Reference examples 1 to 5

Isolation of total nucleic acid

In the reference examples 1 to 5 listed below, the binding, washing and elution conditions were chosen in such a way that both DNA and RNA bind to the mineral support and are eluted together.

Using this example, the use of various alcohols (ethanol, isopropanol, butanol) as a binding reagent is illustrated.

Reference example 1

Isolation of total nucleic acid from kidney tissue

Total nucleic acid was isolated from 15 mg kidney tissue (rat) according to standard protocol 4.1. The tissue was - with 400 ul Ll added and homogenized, then 280 ul Bl were added. The first washing step was carried out with Wl, the elution volume was 2 × 50 μl.

Reference example 2

Isolation of total nucleic acid from liver tissue

Total nucleic acid was isolated from 7 mg liver tissue (rat) according to standard protocol 4.1. The tissue was mixed with 300 ul L2 and homogenized, then 200 ul B2 was added. The first washing step was carried out with Wl, the elution volume was 2 × 50 μl.

Reference example 3

Isolation of total nucleic acid from HeLa cells

Total nucleic acid was isolated from 1 x 10 ⁶ HeLa cells according to standard protocol 4.1. The cells were mixed with 400 ul L2 and homogenized, then 200 ul Bl was added. The first washing step was carried out with Wl, the elution volume was 1 × 50 μl.

Reference example 4

Isolation of total nucleic acid from plasma

Total nucleic acid from plasma was isolated in parallel according to standard protocol 4.1 and 4.2. For this purpose, 200 μl plasma was mixed with 800 μl L3 and 660 μl B2 and mixed; homogenization was not necessary here. In the batch procedure (4.2), an additional 40 μl silica suspension was added. The first washing step was carried out with W2 in both batches, the elution volume was 2 × 100 μl. example 1

Fractional binding of RNA and DNA with constant GTC concentration and increasing ethanol concentration

The dependence of the RNA / DNA binding on the mineral support with constant GTC and increasing ethanol concentration was shown by lysing 10 mg of kidney tissue in 350 μl L4 per sample batch and adding 350 μl of an ethanol / water mixture per batch to adjust the ethanol concentration was, whose ethanol content was between 20 and 90% ethanol in water. For a further batch, 350 μl of absolute ethanol were added. In the respective batches, this corresponded to binding conditions of constant GTC concentration of 1.75 M and increasing ethanol concentration in the range from 10 to 50% (cf. FIG. 1).

In a first series of experiments, 150,000 cpm of a ³² P-labeled 0.9 kb in vitro transcript was added to the lysates set in this way and the lysate was pipetted onto the mineral support fixed in an spin column. It was centrifuged for 15 seconds at 10,000 rpm in a table centrifuge and the amount of the radioactivity bound to the column and the radioactivity in the column breakthrough was determined by Cherenkov count.

The series of experiments was repeated, with instead of the ³² P-labeled RNA, 150,000 cpm of a ³² P-labeled, linearized pTZ plasmid added by Klenow filling reaction being added.

As shown in FIG. 1, the RNA fraction binds to the mineral support under the described conditions from ethanol concentrations greater than 25%, while the DNA fraction only binds from ethanol concentrations greater than 40%. Examples 2 to 8

Isolation of total RNA

In the following examples, the alcohol / salt mixtures were selected for binding to the mineral carrier (cf. FIG. 1) in such a way that selective RNA binding was achieved.

The binding conditions were matched to the type of digestion material (tissue, cell culture, plants, bacteria).

The examples illustrate the use of GTC, GuHCl or GTC / ethanol mixtures for the lysis of the starting materials. The integrity of the isolated RΝA was verified by Northern blotting or RT-PCR.

The processing of the DNA not bound to the support was dispensed with in these examples. Purification of DNA from the column breakthrough is shown in Example 12. In addition, the DNA can be purified by setting the binding conditions selected in reference examples 1 to 5.

Example 2

Isolation of total RNA from spleen tissue

Total RNA was isolated from 15 mg spleen tissue (mouse) according to standard protocol 4.1. The tissue was mixed with 350 ul L4 and homogenized, then 350 ul B4 was added. The first washing step was carried out with W3, the elution volume was 1 × 50 μl. Example 3

Isolation of total RNA from liver tissue (A)

In this example, an ethanol-containing lysis buffer was used, so that the standard protocol 4.1 was slightly modified.

8 mg liver tissue (rat) were mixed with 700 μl L5 and homogenized. The lysate was pipetted onto the spin column and the standard protocol 4.1 was carried out from step 3). The first washing step was carried out with W3, the elution volume was 1 × 50 μl.

Example 4

Isolation of total RNA from liver tissue (B)

Total RNA was isolated from 15 mg of liver tissue (rat) according to standard protocol 4.1. The tissue was mixed with 300 ul L6 and homogenized, then 175 ul Bl added. The first washing step was carried out with W4, the elution volume was 1 × 50 μl.

Example 5

Isolation of total RNA from HeLa cells

Total RNA was isolated from 1 x 10 ⁷ HeLa cells in parallel according to the standard protocols 4.1 and 4.2. The cells were each mixed with 350 ul L7 and homogenized, then 350 ul B4 were added. In addition, 50 μl silica suspension were added in the batch procedure (4.2). The first washing step was carried out with W3, the elution volume was 1 × 50 μl. Example 6

Isolation of total RNA from tobacco

The standard protocol 4.1 is slightly modified for total RNA isolation from plants. After step 1) of the protocol (lysis), a centrifugation step at 5,000 rpm is inserted in a bench-top centrifuge to remove non-lysed cell components, e.g. Fiber residues to separate. The supernatant is removed, binding reagent is added and further processing is carried out from step 2) according to the standard procedure.

Total PJSTA was isolated from 100 mg tobacco leaves according to standard protocol 4.1 modified for plants. The pulverized cell material was mixed with 600 ul L2 and homogenized, then 350 ul B4 was added. The first washing step was carried out with W3, the elution volume was 1 × 50 μl.

Example 7

Isolation of total RNA from E. coli

To isolate total RNA from bacteria, an additional step is inserted before the standard protocol is carried out in order to lyse the cell walls of the bacteria. For this purpose, the cell pellet is resuspended in 400 μg / ml lysozyme in TE and incubated for 5 min on ice and 10 min at room temperature. Then the standard procedure is followed.

Total RNA was isolated from 1 x 10 ⁹ E. coli according to the standard protocol 4.1 modified for bacteria. The pellet was resuspended in 80 ul 400 ul / ml lysozyme in TE and as above incubated as described. Then 270 ul L2 were added, homogenized and 350 ul B4 added. The first washing step was carried out with W3, the elution volume was 2 × 50 μl.

Example 8

Selective RNA binding through optimization of the wash buffer

As this example shows, DNA contaminations can be removed from the specifically bound RNA by optimizing the wash buffer used in the first washing step (standard protocol 4.1.5).

1 x 10 ⁶ HeLa cells were lysed according to standard protocol 4.1 in 350 μl L4, mixed with 350 μl B4 and bound to the silica support. The samples were then washed in the first washing step with the following washing buffer:

Sample wash buffer

M GTC 25 mM% EthaTRIS / HCl nol pH 7.5

1 0.3 + 5

2 0.6 + 5

3 0.9 + 5

4 0.3 - 5

5 0.6 - 5

6 0.9 - 5

7-12 as 1-6 but 10% EtOH

8-18 as 1-6 but 20% EtOH

R *) 1.75 - 35

*) The sample served as a reference; the composition of the wash buffer corresponded to the binding conditions Tab. 1 Composition of the washing buffer for washing out DNA contamination

The further work steps were carried out according to the standard protocol; the elution volume was 1 x 50 μl.

Half of the eluate was analyzed on a 1.2% formaldehyde gel (cf. FIG. 2). The gel was then as below

"Electrophoretic methods" described treated with RNaseA

(see Fig. 3).

Legend for Figures 2 and 3

2: 1.2% formaldehyde gel for analysis of the eluates from Example 8, binding conditions: 1.75 M GTC, 12.5 m; , Nacitrate pH 7.5, 35% ethanol; Washing conditions: cf. Tab. 1. The names of the tracks correspond to the names of the samples in Table 1.

Fig. 3: RNase duration of the gel Fig. 2

Examples 9 and 10

Isolation of DNA

In Examples 9 and 10 listed below, the binding conditions were chosen so that only the DNA can bind to the mineral carrier while the RNA breaks through.

The processing of the RNA not bound to the support was dispensed with in these examples. The purification of RNA from the column breakthrough is shown in Example 11. In addition, the RNA in the column breakthrough can be purified by setting the binding conditions chosen in Examples 2 to 8. The selective DNA binding takes place in the lysis buffer in the absence of alcohol, ie step 2) of the standard protocols 4.1 and 4.2 is omitted.

Example 9

Isolation of genomic DNA from kidney tissue

10 mg kidney tissue (rat) was lysed in 700 μl L8. The DNA was bound to the mineral carrier without the addition of binding reagent and washed in the first washing step with 700 μl L8. The standard protocol 4.1 was then carried out from step 6). The elution volume was 2 x 50 μl.

Example 10

Isolation of genomic DNA from HeLa cells

1 x 10 ⁷ HeLa cells were lysed in 700 ul L9. The DNA was bound to the mineral support without the addition of binding reagent and washed with 700 μl L9 in the first washing step. The standard protocol 4.1 was then carried out from step 6). The elution volume was 2 x 50 μl.

Examples 11 to 13

Separation of total RNA and genomic DNA

The following Examples 11 to 13 for the separate processing of RNA and DNA from the same cell lysate represent links of the above-mentioned examples for isolating RNA, DNA or total nucleic acid.

The separation can take place either by differential binding or by fractional elution of RNA and DNA. Examples 11 and 12

Separation of total RNA and genomic DNA by differential binding

There are two options for differential binding:

After lysis, the conditions can either be selected so that the DNA first binds to the mineral support (Example 11), or the RNA can be adsorbed in the first binding step while the DNA is being processed from the breakthrough (example 12).

Example 11

Isolation of genomic DNA and total RNA from kidney tissue

10 mg of kidney tissue (rat) were lysed in 350 μl L8 and the DNA bound to the mineral carrier in the lysis buffer. 350 μl of B4 were added to the column breakthrough and the total RNA was isolated as in Example 3.1. The genomic DNA was isolated as in reference example 1.

Example 12

Isolation of total RNA and genomic DNA from lung tissue

The total RNA was isolated from 20 mg of lung tissue (rat) as described in Example 2. The unbound genomic DNA in the column breakthrough was isolated by adding 350 μl of Bl and 350 μl of B5 and binding the DNA to the mineral support as described in standard protocol 4.1. The first washing step was carried out with Wl, the elution volume was 2 × 50 μl. Example 13

Separation of total RNA and genomic DNA by fractional elution

The following example illustrates the selective elution of the DNA fraction of the total nucleic acid bound to the mineral carrier.

The binding conditions are chosen so that the total nucleic acid binds to the mineral carrier. The DNA fraction is then eluted while the RNA fraction remains bound. The eluted DNA is bound to a further mineral support and worked up by adjusting it again to DNA binding conditions (cf. FIG. 1).

Isolation of genomic DNA and total RNA from liver tissue

15 mg liver tissue (pig) were lysed in 300 μl L2 according to standard protocol 4.1 1) to 4), 250 μl Bl were added and the total nucleic acid was bound to the mineral carrier. The DNA fraction was eluted with 300 μl W5, while the carrier material was treated with the still bound RNA fraction from 5) according to standard protocol 4.1. The DNA fraction was isolated from the eluate by adding 350 .mu.l of Bl and 250 .mu.l of B5 and binding to another mineral carrier according to the standard protocol 4.1.

Claims

Approaches to the separation of double and single-stranded nucleic acids from sources containing these substances, a sample being treated with at least one mineral carrier, wherein

1.1 the treatment conditions are set by an aqueous mixture of salts and substances containing alcohol groups such that predominantly the single-stranded nucleic acid-containing fraction is adsorbed on a first mineral support, while the double-stranded nucleic acid is not adsorbed, whereupon the single-stranded adsorbed on the first mineral support Nucleic acid, after washing steps which may have been carried out, is eluted under conditions of low ionic strength or with water, and for the non-adsorbed double-stranded nucleic acid which has been collected, conditions are then set by means of a corresponding aqueous mixture of salts and substances containing alcohol groups such that these in particular adsorbable on a second mineral carrier and, after washing steps which may have been carried out, can be eluted from the second mineral carrier under conditions of low Io strength or with water

or 1.2 the treatment conditions are set such that alkaline earth ion complexing substances are present in the absence of substances containing alcohol groups, the single-stranded nucleic acid is not adsorbed on a first mineral carrier under these treatment conditions and is separated from the rest of the sample However, the double-stranded nucleic acid predominantly binds to the first mineral carrier, whereupon the single-stranded nucleic acid, after washing steps which may have been carried out, is eluted under conditions of low ionic strength or with water and for the non-adsorbed single-stranded nucleic acid subsequently by adding alcohol-containing substances Conditions are set so that the single-stranded nucleic acid can then be adsorbed in particular on a second mineral carrier and, after washing steps which may have been carried out, by the second miner Alic sluggish is elutable under conditions of low ionic strength or with water

or

1.3 the treatment conditions are set such that wetting agents, detergents or dispersants are present in the solution in the absence of alcohol-containing substances and the single-stranded nucleic acid is not adsorbed on a first mineral carrier under these treatment conditions and is separated from the rest of the sample However, the double-stranded nucleic acid predominantly binds to the first mineral carrier, whereupon the double-stranded nucleic acid, after washing steps which may have been carried out, is eluted from the first mineral carrier under conditions of low ionic strength or with water and for the non-ad- sorbed collected single-stranded nucleic acid are then adjusted by adding substances containing alcohol groups, so that the single-stranded nucleic acid is then adsorbable in particular onto a second mineral carrier and, after washing steps which may have been carried out, becomes elutable under conditions of low ionic strength or with water

or

1.4 the treatment conditions are adjusted by a corresponding aqueous mixture of salts and substances containing alcohol groups such that the total nucleic acid (single or double-stranded) is adsorbed on a first mineral carrier, followed by fractionation of the single / double-stranded bound to the carrier Nucleic acids by elution of the single / double-stranded nucleic acids from the first mineral carrier by selective elution of the double-stranded nucleic acid by treatment with a solution of reduced ionic strength and concentration of a substance containing alcohol groups or elution of the single-stranded nucleic acid by means of a solution containing an alkaline earth metal complexing substance and / or a wetting, washing or dispersing agent and one or more types of salt,

the other nucleic acid remaining bound to the first mineral carrier and, after washing steps which may have been carried out, being eluted from the first mineral carrier under conditions of low ionic strength or with water and for the double- or single-stranded nucleic acid previously eluted from the first mineral carrier then by increasing the ionic strength or. Concentra tion alcohol-containing substances treatment conditions are set which can lead in particular to the adsorption of the double / single-stranded nucleic acid on a second mineral carrier and, after washing steps which may have been carried out, can be eluted under conditions of low ionic strength or with water.

2. The method according to claim 1, wherein the system for lysing the sources containing the nucleic acids contains chaotropic substances in a concentration of 0.1 to 10 M.

3. The method according to claim 1 and / or 2, wherein the source containing nucleic acids is mixed with a solution containing sodium perchlorate, guanidinium hydrochloride, guanidinium isothiocyanate guanidinium thiocyanate, sodium iodide, potassium iodide and / or combinations thereof Concentration of 0.1 to 10 M contains.

4. The method according to at least one of claims 1 to 3, wherein the nucleic acid-containing source is mixed with a solution containing sodium chloride, lithium chloride, potassium chloride, sodium acetate, magnesium chloride, urea and / or combinations thereof in a concentration of 0.1 to 10 sts included.

5. The method according to at least one of claims 1 to 4, wherein the alcohol group-containing substances are lower aliphatic alcohols such as methanol, ethanol, isopropanol, butanol and pentanol in a concentration of 1 to 90 vol .-% and the salts such as NaCl, KC1 , LiCl, MgCl ₂ , NaAc, in a concentration of 1 to 10 M.

6. The method according to at least one of claims 1 to 5, wherein the mineral carrier made of porous or non-porous metal oxides or mixed metal oxides, silica gel, materials consisting predominantly of glass, such as unmodified glass particles, glass powder, quartz, aluminum oxide, zeolites, Titanium dioxide, zirconium dioxide is built up with a particle size of the mineral carrier material of 0.1 μm to 1,000 μm and a pore size of 2 to 1,000 μm, and the porous or non-porous carrier material can be in the form of loose beds or the carrier materials are designed as filter layers are in the form of filter layers made of glass, quartz or ceramic and / or a membrane in which silica gel is arranged and / or are present as particles or fibers from mineral supports and fabrics made of quartz or glass wool and latex particles with or without functional groups are or Frit materials made of polyethylene, polypropylene, polyvinylidene fluoride, especially ultra high molecular weight polyethylene, high density polyethylene.

7. The method according to at least one of claims 1 to 6, wherein the biological source cell cultures, tissues of all kinds, body fluids such as blood, plasma, serum, urine, faeces, microorganisms such as bacteria, viruses such as cytomegalovirus, HIV, hepatitis B , Hepatitis C, hepatitis δ virus, plants, parts of plants, embryos, seedlings, fruits or the sample containing nucleic acids are mixtures which after enzymatic reactions such as in vitro transcription and / or cDNA synthesis and / or reverse transcription with subsequent PCR attack.

8. The method according to at least one of claims 1 to 7, wherein the cells are first digested (lysed) in an aqueous lysis system containing chaotropic substances or other salts or mixed with them.

9. The method according to at least one of claims 1 to 8, wherein each of the fractions obtained is purified by further chromatographic steps.

10. The method according to at least one of claims 1 to 9, wherein the alkaline earth metal binding substance ethylenediaminetetraacetic acid (EDTA) or EGTA and the wetting, washing or dispersing agent is a sarcosinate.

11. The method according to at least one of claims 1 to 10, wherein the single-stranded nucleic acid is RNA and the double-stranded nucleic acid is DNA.

12. An aqueous solution containing 0.5 to 3.0 M guanidinium thiocyanate and / or guanidine hydrochloride, 1 to 50% ethanol and / or isopropanol.

13. Use of the solution according to claim 12 as a buffer for the lysis of nucleic acid-containing sources and / or binding of nucleic acids to mineral carriers.

14. An aqueous solution containing 0.1 to 3 M guanidine thiocyanate and / or guanidine hydrochloride, 1 to 30% ethanol and / or isopropanol.

15. Use of the solution according to claim 14 as a buffer for washing out of nucleic acids bound to mineral carriers.

16. An aqueous solution containing 1 to 5 M guanidine thiocyanate and / or 1 to 8 M guanidine hydrochloride, 0.1 to 3% sarcosyl.

17. Use of the solution according to claim 16 as a buffer for binding DNA to mineral carriers.

18. Use of a solution containing 0.5 to 8.0 M guanidium thiocyanate and / or guanidinium hydrochloride, 1 to 50% ethanol and / or isopropanol.

19. An aqueous solution containing 1 to 5 M guanidinium thiocyanate and / or 1 to 8 M guanidinium hydrochloride, 5 mM to 200 mM EDTA or EGTA.

20. Use of the solution according to claim 19 as a buffer for binding DNA, double-stranded nucleic acid to mineral carrier.

21. Use of the solution according to claim 19 as a buffer in order not to bind single-stranded nucleic acid (RNA) to mineral carriers.

22. Kit for carrying out the method according to at least one of claims 1 to 11 with mineral carrier, which is arranged in a hollow body suitable for flow, solutions according to one of claims 12, 14, 16 and / or 19 and further auxiliaries and / or accessories .

23. Use of the nucleic acids isolated by the method in amplification reactions such as PCR, LCR, NASBA or 3SR for medical diagnostics.

24. Use of the nucleic acids isolated by the method for medical diagnostics without using amplification reactions.